Reactivation of Tumor Suppressor Genes in Breast Cancer by Dietary Supplements Hypermethylation-silenced tumor suppressor gene (TSG) is a hallmark of most cancers, including breast cancer and has been detected in pre-invasive lesions and/or high-risk tissues. In contrast to genetic mutation, DNA methylation is susceptible to change and reversible;therefore, it represents an excellent target to develop DNA methylation inhibitors for chemotherapy and chemoprevention. Reactivation of TSGs by azanucleoside DNA methylation inhibitors, such as decitabine and 5-azacitidine will result in restoration of their biological functions and represents a novel, superior chemotherapeutic strategy for treatment of various leukemia, including myelodysplastic syndrome (MDS) compared to cytotoxic agents. However, there are limitations in developing azanucleosides as cancer prevention agents because of their chemical instability and high toxicity. Therefore, there is a surging interest to identify DNA methylation inhibitors from dietary supplements, e.g. EGCG to reactivate TSGs in cancers as chemopreventive agents because of their pharmacological safety in long-term use. Using an integrated platform consisting of the DNMT1 homology modeling, in-vitro cell-free M. SssI assay and a rapid, robust, specific and sensitive LC-MS/MS method for determination of both global and specific promoter DNA methylation levels established on our group, parthenolide and curcumin have been found to possess DNA hypomethylation activity. Curcumin, the active constituent of turmeric and wide-consumed dietary additives, is significantly more potent (>100 fold) than parthenolide as a DNA methylation inhibitor with an IC50 of 30 nM on M. SssI and 35-40% global DNA hypomethylation at 0.1 5M in MCF-7 cells. This data suggests that curcumin may be a potent and safe DNA methylation inhibitor. Curcumin has been shown strong antiproliferation effects on lung, breast, prostate and colon cancers through multiple molecular targets and signaling pathways and extensively tested as a cancer chemoprotective agent. Therefore, two tightly associated aims are pursued in this proposal to elucidate the underlying molecular mechanism of its hypomethylation activity and test our hypothesis whether curcumin can hypomethylate and reactivate TSGs involved in initiation, propagation, and metastasis of breast cancer. The first aim is to further evaluate the hypomethylation activity of curcumin in-vitro and in- vivo for induction of promoter hypomethylation of specific TSGs e.g. RASSF1A and their reactivation in breast cancer cell lines at its attainable in-vivo concentration. The second aim is to elucidate the other underlying molecular mechanism of the hypomethylating activity of curcumin in breast cancer cell lines. The proposed studies will yield data which will steer the future development of curcumin as a chemopreventive agent in breast cancer. PUBLIC HEALTH RELEVANCE: The project will establish curcumin as an effective DNA methylation inhibitor to reactivate several tumor suppressor genes involved in cancer cell apoptosis, cell cycle control and metastasis in vitro and in vivo and elucidate its underlying molecular mechanism. It is expected that the result will provide a direct molecular mechanism of curcumin and steer its future use as a chemopreventive agent on breast cancer and potentially for other prevalent cancers e.g. colon cancers.